1. Field of the Invention
The present invention relates to a cell culture support for stable mass production of cell sheets.
2. Background Art
A cell sheet is a sheet-form cell aggregate, in which cells are connected via intercellular junction at least in a single layer. Cell sheets are used in regenerative medicine and the like. A cell sheet can be obtained by culturing cells on a cell culture support such as a Petri dish. However, a cell sheet formed on a cell culture support is tightly adhered to the surface of the cell culture support via adhesion molecules and the like. Therefore, it is not easy to detach a cell sheet from a cell culture support rapidly without breaking the cell-to-cell junctions.
Under the circumstances, studies have been made on methods for efficiently detaching a cell sheet from a cell culture support. The detaching methods can be classified into two groups. In the first method, the interaction between a cell culture support and cells is weakened by an enzymatic reaction. In the second method, a cell culture support whose cell adhesive ability is weak or can vary is used.
To explain more specifically, in the first method, enzymes such as protease (proteolysis enzyme) and collagenase (collagenolytic enzyme) are used to degrade proteins constituting intercellular adhesion molecules (involved in tight junction, adherens junction, desmosome junction, gap junction and hemi desmosome junction), collagen matrix surrounding a culture and the extracellular matrix (ECM) formed between cells and a cell culture support. In this method, not only the interaction between cells and the surface of a cell culture support but also cell-cell junctions are weakened. The method has long been used in the field of cell culture. Since the binding substances which are degraded by the method are those produced in cells, tissues and organs to be cultured, they can be regenerated in certain conditions and period of time after a cell sheet is detached.
Nevertheless, the first method has problems. It requires a long time to regenerate the binding substances. Moreover, the cell sheet formed by the method is more or less damaged. Thus, the method is not suitable as a method for manufacturing a cell sheet for use in regenerative medicine.
Under these circumstances, the second method, which uses a cell culture support whose cell adhesive ability is weak or can vary, has been newly developed.
Examples of such cell culture support whose cell adhesive ability is weak include the supports disclosed in Patent Document 1 and Patent Document 2. These documents disclose technique with which cells are cultured on a cell culture support having fine pillars called nanopillars on the surface. With this technology, the cell culture support and a culture material are in contact with each other only at an extremely small area. Therefore, it is believed easy to detach and recover a cell sheet with little damage to the cell sheet.
Nevertheless, as is described in Non-Patent Documents 1 and 2, cell adhesion and the behavior of cells adhered differ depending on whether the cells are adhered to a flat surface or an irregular surface. Culturing on the nanopillars has such problems that adhesion of cells and extension of a cell sheet are delayed, and pseudopoium is formed from a cell surface. Moreover, cells enter the depressions when the depressions of the cell culture support have a width of 20 μm or more.
Examples of a cell culture support whose cell adhesion ability can vary include a cell culture support having a coating of a temperature responsive polymer on the cell proliferation surface thereof (Patent Document 3). A temperature responsive polymer is most preferably used for the purpose of altering cell adhesion ability. Besides this, however, a pH responsive polymer and an ion responsive polymer can also be used. Patent Documents 1 and 2 describe that a temperature responsive polymer is used in combination with culture using nanopillars. The use of a temperature responsive polymer in cell culture is also mentioned in Patent Document 4.
As a method for manufacturing a temperature responsive polymer layer, Patent Document 3 describes a graft polymerization method, in which a polymerization reaction of monomers by electron beam irradiation and a reaction to immobilize (graft) a temperature responsive polymer to a substrate surface by covalently binding at least one end of the temperature responsive polymer to the molecule constituting the substrate are performed. Nevertheless, as is described in Patent Document 4, a temperature responsive polymer layer formed by the graft polymerization method had a problem that it exhibited temperature responsiveness only when a ratio of materials used and conditions for electron beam irradiation are constant.
Patent Document 4 discloses that the amount of a solvent is determined to lower the amount of the residual solvent in a material monomer/solvent mixture to be coated onto a substrate surface before electron beam irradiation. However, in the methods described in Examples and Comparative Examples in Patent Document 4, complicated process control was required for ensuring reproducibility in the amount of the residual solvent, such as drying under natural condition in a constant temperature and humidity chamber, drying under a nitrogen gas stream, vacuum drying and drying by heating within the range not affecting the polymerization of residual monomers. In addition, in Examples and Comparative Examples in Patent Document 4, a solvent whose boiling temperature is 120° C. or lower is used as a solvent to dissolve the coating material for the purpose of improving the polymerization efficiency by electron beam energy. When a composition in which a monomer material is dissolved into such solvent is deposited on the dish to coat, there was a problem that the monomer tended to crystallize. Existence of microcrystals was confirmed by microscopic observation even when no crystal is visible to the naked eye.
The present inventors conducted studies and thus found a problem that a sufficient amount of a temperature responsive polymer is not coated, since polymerization is inhibited in crystal regions when the film with monomer crystals is irradiated with an electron beam. To use the temperature responsive polymer, only the grafted polymers are exposed by washing away the excess free polymers after the graft polymerization by radiation. It is assumed to be effective to reduce the amount of the material used for coating to reduce irregularity of the coated surface and shorten the washing time. However, reducing the amount of the material used for coating was problematic because the reduction would result in promoting crystallization.
The present inventors possess many technologies in the field of printed phase technology with the use of materials for electron beam, such as anti-fog film and coated plywood board. The present inventors have found that a film to which a polymerizable oligomer and a prepolymer are added has excellent decorative coating properties for printing, coating and adhesive hardening by electron beam irradiation (Patent Document 5).
When a temperature responsive polymer is grafted on a substrate surface with the use of radiation, the conditions for each radiation are determined to obtain a uniform distribution of the polymer to be grafted on the substrate. Nevertheless, there were cases where the detachment of the cell sheet did not proceed smoothly depending on the material constituting the substrate, even though the polymer was successfully immobilized onto the substrate surface and a hydrophilic/hydrophobic transition was induced by the aforementioned polymer.
On the other hand, Patent Document 15 discloses a method of multilayered culture for epithelial type cells, in which, when epithelial type cells are cultured on a porous membrane for a cell culture insert, both of the upper layer and the lower layer divided by the porous membrane are filled with a culture medium, and the culture medium is constantly supplied from the lower layer to the cells through the porous membrane while the cells are cultured. In addition, Patent Document 15 also discloses that a temperature responsive polymer is deposited on the aforementioned porous membrane. The cell culture insert disclosed in Patent Document 15 enables a cell culture on a permeable porous membrane, in which the components of the liquid culture medium are diffused to both luminal and basal sides of the cells, similarly to the in vivo process, in an attempt to achieve an in vitro cell culture which is physiologically similar to the in vivo environment. By using the cell culture insert, it is made possible to perform a three-dimensional culture, co-culture for 2 kinds of cells, cell migration/infiltration assay using cells passing through a porous membrane, drug penetration assay using a drug passing through a porous membrane and the like. The cell sheet being cultured by a release culture with the use of a feeder cell and cell growth factor on the undersurface of a cell culture insert can provide more multilayered cells and, in addition, more consistent vertical orientation than the ones being cultured without a feeder or by directly filling a Petri dish with a culture medium containing a cell growth factor. For example, corneal epithelial cells form a multilayer with their basal side down, and mucosal cells are oriented to have their microvilli up.
Patent Document 17 describes that in relation to cardiac muscle treatment using a cell sheet, the cardiac muscle cultured on a substrate to which a temperature responsive polymer is disposed is detached as a cell sheet, and can be used as a transplantable cardiac muscle cell sheet for improving the cardiac function and suppressing cardiac deformation.
Non-Patent Document 6 describes that the cardiac muscle cell sheet manufactured by this method exhibits autonomous pulsating and that when the cell sheets are bilayered, they are adhered with each other with the ECM without sutures, and the bilayered sheet exhibits a synchronously pulsating over a short period of time.
However, it is known that the autonomously pulsating cell sheet manufactured by this method has a random pulse orientation and, thus, if used for treating cardiac muscle, the cell sheet adjusts its orientation to that of a heart receiving transplantation of the cell sheet, which has oriented pulsating, when transplanted to the heart, and the cardiac muscle cell sheet itself adjusts its orientation to the expansion and contraction orientation by repeating expansion and contraction by adding an external force in the culture medium.
However, problems occurred when a cell sheet detached from a substrate was maintained in a culture medium until the cardiac muscle cell sheet itself acquired its own expansion and contraction orientation. The ECM of the sheet was eliminated (digested), and rapid adhesion at the time of transplantation and synchronization of expansion and contraction of the pulse were difficult to take place.    Patent Document 1: JP Patent Publication (Kokai) No. 2004-170935 A    Patent Document 2: JP Patent Publication (Kokai) No. 2005-168494 A    Patent Document 3: JP Patent Publication (Kokoku) No. 6-104061 A (1994)    Patent Document 4: JP Patent Publication (Kokai) No. 5-192130 A (1993)    Patent Document 5: JP Patent No. 2856862    Patent Document 6: JP Patent No. 312660    Patent Document 7: JP Patent No. 3491917    Patent Document 8: JP Patent Publication (Kokai) No. 9-12651 A (1997)    Patent Document 9: JP Patent Publication (Kokai) No. 10-248557 A (1998)    Patent Document 10: JP Patent Publication (Kokai) No. 11-349643 A (1999)    Patent Document 11: JP Patent Publication (Kokai) No. 2001-329183 A    Patent Document 12: JP Patent Publication (Kokai) No. 2002-18270 A    Patent Document 13: JP Patent Publication (Kokai) No. 5-244938 A (1993)    Patent Document 14: International Patent Application No. WO 01/068799    Patent Document 15: JP Patent Publication (Kokai) No. 2005-130838 A    Patent Document 16: JP Patent Publication (Kokai) No. 2006-320304 A    Patent Document 17: JP Patent Publication (Kokai) No. 2003-306434 A    Non-Patent Document 1: M. J. Dalby et al., Biomaterials, 25, 5415-5422 (2004)    Non-Patent Document 2: C. C. Berry et al., Biomaterials, 25, 5781-5788 (2004)    Non-Patent Document 3: W. D. Snyder et al., J. Am. Chem. Soc., 97, 4999 (1967)    Non-Patent Document 4: L. F. Fieser et al., “Reagents for Organic Synthesis,” John Wiley & Sons, New York, N.Y., 1967    Non-Patent Document 5: O. H. Kwon, J. Biomed. Mater. Res., April (2000); 50 (1): 82-9    Non-Patent Document 6: Y. Haraguchi et al., Biomaterials, 27, 4765-4774 (2006)